JP2022517568A - A device that quantifies biological components dispersed in a fluid - Google Patents

Abstract

A device 100 for quantifying biological components 3, 3', 3'' in a fluid, wherein the detection electrodes 4, 4', 5, 5', 6, 6', 34, 34', 84, 84', Measurement cell 1 with 94, 94', 184, 184', 194, 194' and reference electrodes 7, 7', 8, 8', and input signal generation, impedimetric measurement, output signal amplification, and user. Electronic unit 201 for communication with the interface and means 101, 102, 103 for generating a magnetic field with a corresponding gradient that can be modulated in time, said magnetizing means stored in the measurement cell. Separation of components 3, 3', 3'' to be quantified from the rest of the solution, and detection electrode 4 coupled with concentrators 10, 10', 10'', 14, 14', 14'', 15. A magnetic field capable of causing their concentration on 4,', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194'. Device 100 with means 101, 102, 103 configured to generate.

Description

The present invention is biological, cellular, or acellular particles dispersed in a sample of a biological fluid (blood, urine, saliva, sweat, etc.) or a fluid sample extracted from a biological solid substance (feces, etc.). It relates to an apparatus for quantifying (quantifying) a component.

This quantification is performed by separating the components of interest from the rest of the sample by magnetophoresis, concentrating (aggregating) them, and detecting the amount of these components by impedimetry. For the purposes of this specification, "cell biocomponent" or simply "cell component" means a biobody that is larger or equivalent in size than a cell, eg, blood cells (erythrocytes, leukocytes, platelets, etc.), urine. Examples include small bodies contained in, pathogens such as bacteria, and eggs of specific parasites. A "non-cellular biological component" or simply a "non-cellular component" is understood to be a living body-derived body that is smaller in volume than a cell and larger in volume than an individual molecule. Such non-cellular components can be crystals of specific substances expressed under specific pathological conditions, such as hemozoin crystals produced in red blood cells infected with Plasmodium malaria.

Usually, the apparatus of the present invention aims to quantify all biological components dispersed in a fluid having various magnetic properties depending on the medium in which they are suspended. For the purposes of the present specification, this category is said to have biological components exhibiting unique magnetic behavior that allows electrophoretic separation from the counterpart under physiological conditions, eg, after certain transformations due to pathological conditions. It is stipulated. More specifically, the present invention is one or more cells and non-cells dispersed in a fluid extracted from a biological fluid (blood, urine, sweat, saliva, etc.) or a solid biological sample (feces, etc.). It relates to a device that makes it possible to spatially separate and concentrate a biological component by utilizing the difference between the magnetic property of the component and the magnetic property of another non-target component, and foresee. After separation and concentration, the apparatus of the present invention measures the impedance change between two or more electrodes placed close to the concentration zone to obtain such cellular and non-cellular biological components. It is believed that quantification will take place.

As an example, one of the application areas of the apparatus of the present invention can be implemented and realized in a manner such as that used for the detection and quantification of parasite eggs (eg, cystosoma mansoni) in infected feces. In this case, these eggs have paramagnetic behaviors that differ from the typical diamagnetic behaviors of aqueous solutions capable of resuspending them (S. Karl et al., PLOS Infected Tropical Diseases, 7 (5)). , 2219 (2013)).

Another area of application of the invention is for all pathologies that cause a modified form of the magnetic properties of one or more types of blood cell components and / or cause the formation of substances with magnetic properties different from plasma. For diagnostic purposes, the substance is absent under physiological conditions or is present in different concentrations. In particular, as a pathological condition that causes a deformed form of the magnetic properties of erythrocytes, an increase in methemoglobinemia due to malaria or poisoning is known. For example, in the case of malaria, Plasmodium malaria, which is the pathogen of malaria, is known to produce a specific substance called hemozoin, which is a paramagnetic substance, as described above. In particular, hemozoin is produced in the form of crystals that accumulate in infected erythrocytes and exhibits paramagnetism with respect to the medium in which the crystals are dispersed, namely plasma. In addition, in the non-early stages of malaria, the membrane of infected red blood cells is destroyed and hemozoin crystals are released into plasma, which are instead diamagnetic. Furthermore, it is also known that the magnetic properties of cellular components in blood do not change, but on the contrary, their densities change. Sickle cell anemia is an example of this type, where the density changes even if the diamagnetism of the red blood cells does not change. In this case, for example, by adding a strong magnetic substance such as gadolinium to plasma, the magnetic difference between erythrocytes and the gadolinium solution added to plasma and the difference in density between morbid erythrocytes and healthy erythrocytes can be obtained. It may be conceivable to utilize and perform isolation and thus count pathogenic red blood cells.

At the cutting edge, techniques for separating cellular components in blood are known, which are based on various magnetic behaviors of cellular components under physiological and pathological conditions. In particular, US Pat. No. 5,985,153 describes a device for the separation of cells or other magnetically sensitive biological entities, including a substrate, an external magnetic field generator, and a microfluidic system for blood uptake and removal. Has been done. Instead, US Pat. No. 0127222 aims to immobilize cells pre-marked with magnetic particles so that they can be attracted by a ferromagnetic structure formed on the chip and placed in an external magnetic field. General-purpose system is described. International Publication No. 2010/091874 describes a particular ferromagnetic structure composed of magnetic tubes, which can attract magnetic particles to a particular location where the domain wall is located. All the prior art documents mentioned above and some of the scientific literature described in the bibliography "S. Bakdi et al., Optimized high magnetic separation for isolation for Isolation of Plasmodium-infected red blood cell 20 years. , 38 ”,“ J. Nam et al., Magnetic Separation of Malaria-Infected Red Blood Cells in Various Developental Stages, Anal. Chem., pp. 85, 7316-73 Bruno Frazier, Paramagnetic catalog mode magnetophoretic microseparator for high efficacy blood cell separations, Lab Chip, 266-27 It has been described and no mention is made of detecting the number of such components.

U.S. Pat. - pp. 3909 "and" M., et al. Ibrahim, J. Claudel, D. Kourtiche and M. Nadi, Geometric parameters optimization of planar interdigitated electrodes for bioimpedance spectroscopy, J Electr Bioimp, vol.4, 13 ~ 22 pages, "2013" instead describes techniques for impedimetric quantification of cellular components. However, these techniques have never been used in combination with magnetic separation and concentration. Impedimetric detection requires that the volume fraction of the components near the electrodes be sufficiently high to obtain a signal-to-noise ratio of the output signal sufficient to ensure correct quantification of the separated components. This concentration is usually obtained using a microfluidic technique aimed at the movement of blood fluid, which significantly increases the complexity of the system and is suitable for use by non-expert users, such as the patient himself. Is not suitable. The proposed device attempts to overcome these difficulties by magnetically concentrating and separating the components of interest in the area of the measurement cell where the detection electrodes are located, instead of separating by motion of the microfluidics. It is something to do.

Also, for the diagnosis of all pathologies that cause changes in the magnetic properties of one or more types of blood components, a non-expert user has just collected, arbitrarily diluted, and anticoagulant to make measurements. Blood treated with a coagulant is dropped onto a special support of the cell and then brought into contact with the cell substrate in which the concentrator element and electrodes are housed (contained), this time the component of gravity is directed towards the concentrator. It can be placed in an external magnetic field to counteract the magnetic attraction. The measurement cell can also be a pre-assembled measurement cell with the substrate and support placed at appropriate distances, optionally with pre-administration fluid that only performs blood dilution and anticoagulation and moves within the measurement cell. It can also be integrated with a microfluidic system that has. The measurement cell of the apparatus of the present invention is solely the function of sample dilution and transfer, even if this embodiment is more complex than the embodiment that predicts that separation will be performed only by magnetophoresis and impedimetric detection. It is clear that it can be further integrated with microfluidic systems that perform functional blood movements for the same separation of components. Assuming that when the blood collection volume is about 10 microliters and the maximum distance from the concentrator is 20 to 200 micrometers and the capture of the target component is performed, the size of the effective area for capture on the substrate is. It should be about 1 cm ² and in particular 0.1-5 cm ² . The support and therefore the measuring cell must be about the same size. With such an effective area value, a high concentration of the target component is required in order to secure a sufficient signal-to-noise ratio. As explained more clearly here, this concentration traps droplets containing the component to be quantified, assuming that the components do not slide parallel to the substrate and all components are captured. It can be quantified by the so _- called concentration coefficient FC given by the ratio of the effective area of the substrate to the area defined by the detection electrode. In order to sufficiently secure the signal-to _- noise ratio of the output signal, the concentration coefficient FC is preferably at least about 100.

U.S. Pat. No. 5,985,153 U.S. Pat. No. 0127222 International Publication No. 2010/091874 U.S. Patent Application Publication No. 2012/0003687

S. Karl et al. , PLOS Selected Tropical Devices, 7 (5), p. 2219 (2013) S. Bhakdi et al. , Optimized high gradient separation for isolation of Plasmodium-infected red blood cells, Malaria Journal 2010, 9, 38 J. Nam et al. , Magnetic Separation of Malaria-Infected Red Blood Cells in Various Developmental Stages, Anal. Chem. , 85, pp. 7316-7323 (2013) Ki-Ho Han and A. Bruno Frazier, Paramagnetic capital mode magnetophoretic microseparator for high efficiency blood cell separations, Lab Chip, pp. 6, 265-27 E. Du. , Et al. , Electrical Impedance Microflow Cytometry for characterization of Cell Disease States, Lab Chip. October 7, 2013, 13 (19), pp. 3903-3909 M. , Et al. Ibrahim, J. Mol. Claudel, D.M. Kourtiche and M.K. Nadi, Geometry parameters optimization of planar interdictated electrodes for bioimpedance spectroscopy, J Electroimp, vol. 4, pp. 13-22, 2013 M. Giacometti et al. APPLIED PHYSICS LETTERS 113, 203703 (2018) K. Hand and A. B. Frazier, J.M. Apple. Phys. 96 (10), p. 5797 (2004)

Therefore, an object of the present invention is to be able to quantify the target component dispersed in a liquid of 5 to 500 μL (5 to 50 μL in the case of blood), and a biological cell component up to several tens of components per 1 μL. It is an object of the present invention to provide an apparatus for generating an output signal having a signal-to-noise ratio so that non-cellular components can be detected at a lower limit concentration.

This object is achieved by the apparatus of the present invention, which is:
-A measurement cell with a detection electrode and a reference electrode,
-Electronic units that generate input signals, make impedimetric measurements, amplify output signals, and communicate with user interfaces.
-A means of generating a magnetic field with an appropriate gradient that can be modulated over time, said magnetizing means, in combination with a concentrator stored in a measuring cell, the rest of the solution to be quantified components. It has means that are configured to generate a magnetic field that can be separated from and concentrated on the detection electrode.

The apparatus of the present invention can further have a microfluidic system with a pre-administration fluid that performs dilution of the biofluid sample and movement within the measurement cell. For this purpose, in turn, the microfluidic system,
-Means for collecting fluid samples in cartridges and diluting with premedicated fluids,
-Has a means for moving the diluted fluid sample into the measurement cell.

This time, the measurement cell is
-Multiple detection electrodes and
-At least one concentrator, wherein the at least one concentrator is configured to magnetically attract the component to be quantified and concentrate the component on the detection electrode, and the detection electrode is the at least one. With at least one concentrator located in close proximity to the concentrator,
-With at least one reference electrode pair, preferably one reference electrode pair per detection electrode pair, wherein the reference electrode is placed away from the at least one concentrator. ,
-A substrate configured to house a detection electrode, a reference electrode, and a concentrator, wherein the substrate has a first surface facing the outside of the cell and a second surface facing the inside of the cell. With the board
-A support having a first surface facing the outside of the cell and a second surface facing the inside of the cell and facing the second surface of the substrate.
-With at least one spacer element configured to confine the sample and keep the substrate away from the support.
-With a mechanical housing configured to house the cell and form electrical contact between the electrodes,
-Has an electronic board for connecting to an external electronic unit.

The at least one concentrator can be a cylinder, a parallelepiped or other shaped element placed on the substrate and placed on the detection electrode and is made of a ferromagnetic material. The concentrator produces a strong local magnetic field gradient that attracts the component to be quantified, concentrating the component in close proximity to the concentrator and thereby close to the detection electrode. In this way, by appropriately adjusting the sizes of both the concentrator and the detection electrode, the concentration coefficient can be increased to the value required to obtain a sufficient signal-to-noise ratio.

The detection electrode is placed close to the concentrator element, while the reference electrode is placed away from the concentrator element. In other words, the reference electrode is placed in the area without the concentrator. For the purposes of the present specification, "the detection electrode is arranged close to the concentrator" means that the detection electrode is arranged so that the distance in the direction perpendicular to the substrate with respect to the concentrator does not exceed 5 μm. , The projection of the detection electrode on the substrate plane is included in the projection of the concentrator on the substrate plane, or is not included in the projection of the concentrator on the substrate plane, but does not exceed twice the width of the electrode. It means that it does not depart from the projection of the concentrator on the substrate in the direction of the width (eg, maximum dimension) with respect to the value. The phrase "the reference electrode is placed away from the concentrator" is instead the distance in the width direction of the concentrator between the projection of the concentrator on the substrate and the projection of the detection electrode on the substrate. It means that the detection electrode is arranged with respect to the concentrator so that it is equal to at least twice the unique width of.

In this way, the separate components are selectively accumulated on the detection electrode, but not on the reference electrode, so that the impedance between the detection electrodes is higher than the spurious impedance that can be recorded between the reference electrodes. Make certain changes. Therefore, the output signal of the impedimetric quantification system is proportional to the difference between the impedance change recorded between the detection electrodes and the impedance change recorded between the reference electrodes. From this output signal, the number of components of interest, or similarly, the concentration can be estimated by comparing it with an appropriate calibration curve using a processor.

The magnetic field generating means is configured to realize magnetic field and relative gradient modulation in the substrate containing the concentrator element, that is, from a minimum value corresponding to about 10% of the saturated magnetic field of the concentrator. It is possible to create time variation in magnetic field strength up to a maximum value that must be higher than the saturated magnetic field of the concentrator. As will be described in detail later, when the magnetizing means is made of a permanent magnet, this modulation can be simply obtained by a linear motion of approaching / moving away from the substrate of the magnet. In particular, when the magnet is brought close to the substrate, the component is captured, but when the magnet is moved away, the component is desorbed from the concentrator and the electrode. For components with a resistance greater than the resistance of the liquid, the resistance measured at the end of the detection electrode shows a positive increase at the specific time _τC at the approach stage. On the other hand, when the magnet suddenly moves away, the resistance value returns to the initial value at the time of desorption of the component at another peculiar time _τR . Selective capture and release activation by modulation of the magnetic field on the substrate allows for more accurate measurement of component-related impedance changes. In this way, selective capture and release activation makes it easy to identify false variations that are not synchronized with the movement towards / away from the magnet, and more than the impedance change that effectively corresponds to the component. Accurate measurements can be obtained and the background and corresponding drift improved removal can be obtained.

The increased sensitivity of the test associated with an increase in the ratio of the true signal to the signal due to false positives, and the possibility of distinguishing different blood cells, is that the measurement cell is relative to the horizontal plane, i.e. to the plane perpendicular to the gravitational acceleration vector. Further guaranteed by the fact that it can be tilted.

In fact, the separation of components from the matrix in which they are dispersed depends on the characteristics of the components and the gravitational and magnetic attraction in a manner determined by the angle α formed between the gravitational acceleration vector and the line perpendicular to the substrate. Caused by conflict.

As will be apparent in the examples below, the capture efficiency of the captured components varies depending on the angle α formed by the line perpendicular to the substrate and the gravitational acceleration vector. When α = 0 ° (FIG. 3a), that is, when the measuring cell is parallel to the horizontal plane, the magnetic force FM is the weight force (mg) and the _Archimedes force (F _b ), or more generally the buoyancy (F _b ). Because it is antiparallel to the resultant force, a high gradient of the magnetic field created by the magnetic field generating means is required to avoid sedimentation of the components on the support and instead promote upward movement. As shown in FIG. 3b, when α is a value between 0 ° and 90 °, the magnetic force is the resultant force of the weight force and the Archimedes force, more generally the resultant force of the weight force and the buoyancy (F _b ), that is, ( It is necessary to compare only the projection of mg + F _b ) cos α along the perpendicular line n. Increasing the angle α reduces the threshold of magnetic force that causes the capture of a given component beyond that. Therefore, it is possible to select the optimum angle for distinguishing the capture of different components in the sample without affecting the generated magnetic field. In this mode, the component falls down in a chamber arranged at an angle to the vertical, but during its sliding motion, the component is drawn towards the substrate, sensing the magnetic field of the concentrator and therefore. Be captured. An extreme example is obtained when α = 90 ° (FIG. 3c), where the weight force and the magnetic force do not compete and act along the orthogonal axis. The components fall downwards, but at the same time are attracted horizontally towards the substrate by the magnetic field of the magnet. In this configuration, there is no threshold to allow the components with different magnetic moments to be distinguished, and any paramagnetic component is attracted towards the substrate. However, the amount of components that can be captured differs depending on the strength of the attractive force in the horizontal direction that deviates from the falling motion in the vertical direction, so that the capture efficiency changes. For the above reasons, the measurement cell is fixed at an inclined position where the angle α between the line perpendicular to the substrate of the cell and the gravitational acceleration vector is 0 ° to 180 °.

The overall capture efficiency when α> 0 ° is not determined solely by the local capture of components present in the capture volume in close proximity to each concentrator. Gravity-induced downward sliding of components is defined as the number of components captured per unit of liquid volume in which they are suspended, thereby migrating the components closer to the concentrator. , Allows an increase in capture efficiency. By shaping the confinement ring and the geometry of the concentrator and electrodes to geometrically carry the components on the effective capture area during their gravity-induced sliding motion, thereby capture efficiency. Can be further enhanced.

Next, a second object of the present invention is to provide an apparatus for quantifying cellular and non-cellular components capable of distinguishing various components.

For this purpose, the measuring cell can have a variable tilt instead of a fixed tilt, and the device of the present invention is configured to vary this tilt from 0 ° to 180 °. It may have a mechanical system.

In other words, the apparatus of the present invention is configured to vary the angle α formed between the perpendicular to the substrate of the measuring cell in which the fluid sample is collected and the gravitational acceleration vector from 0 ° to 180 °. May have a mechanical system.

In addition, additional information in identifying the properties of the same captured component, even with the same magnetic field time modulation as described above and the natural measurements of capture and release times, which are specific to the component to be quantified. I will provide a.

As mentioned above, the apparatus of the present invention is one of the causes of magnetic variation in one or more biological components among various pathological conditions at the time of diagnosis in which the apparatus of the present invention can be used. Although applicable to the diagnosis of any condition, the diagnostic devices for this type of condition currently on the market include those typical of endemic areas often found in developing countries. Malaria is of particular interest in that it has some limitations that do not necessarily make it easier to use this device in particularly disadvantageous situations. The most sensitive methods currently available for diagnosing malaria are actually based on genetic recognition of various Plasmodium species by PCR (Polymerase Chain Reaction). This type of method is particularly complex and delicate, which makes it difficult to apply in non-technically advanced situations. Also, PCR is not a pan-plasmodium method, but it targets specific races and is therefore exposed to problems due to uninterrupted mutations in Plasmodium. On the other hand, the "thin smear and / or thick droplet" method, which consists of counting erythrocytes infected with Plasmodium in blood droplets under a light microscope, does not require complicated instrumentation, but is advanced. It requires human resources with specialized knowledge, and the interpretation of the results involves some variability and a long analysis time. On the other hand, rapid tests based on antibody-antigen interactions (RDT: Rapid Test) are characterized by low sensitivity to prevent their use for early diagnosis. Moreover, due to the potential presence of antigens in the patient's body in the epidemic area, methods based on antibody-antigen interactions result in numerous false positives.

Therefore, a third object of the present invention, whether pan-plasmodium or not, is sufficiently sensitive and the available economic means do not allow the use of complex instrumentation and professional personnel. It is to provide a device that enables early diagnosis of malaria, which is simple and economical enough to be used in the community.

This object is achieved by the apparatus of the present invention, because the apparatus can perform magnetic separation and quantification of infected red blood cells as well as direct detection of free hemozoin crystals in plasma. Quantification of infected erythrocytes, in some cases, to calculate the ratio of infected erythrocytes to healthy erythrocytes even in the early stages of the disease prior to the completion of the first Plasmodium regenerative cycle (48-72 hours). Allows a direct estimate of malaria, which is usually quantified by. Instead, direct detection of hemozoin crystals is particularly useful in the non-early stages of the disease, for example at the time of the first heat attack, where the red blood cells have already undergone membrane breakage. This is because the only thing that can actually be quantified in the circulation is free hemozoin.

These and additional objects of the invention are described below in some preferred embodiments of the invention according to, along with a non-limiting example of the more general concept set forth in the claims, with examples relating to the experimental tests performed in the present invention. It will become clearer by looking at the embodiments for carrying out the invention of.

Refer to the accompanying drawings for embodiments for carrying out the following inventions.

It is a front view of the details of the device according to the present invention, the details having a measuring cell and a magnetization means. It is a side view of the apparatus by this invention. It is a front view of the details of the apparatus according to the present invention and the vector diagram of the force when α = 0 °, the details having a central portion of a measuring cell and magnetizing means. A front view of the details of the apparatus according to the present invention and a vector diagram of the force when the generic angle α is 0 to 90 °, the details having a central portion of a measuring cell and magnetizing means. It is a front view of the details of the apparatus of the present invention and the vector diagram of the force at α = 90 °, the details having a measuring cell and a magnetization means. It is a good example of the measurement method carried out by the apparatus of the present invention through the modulation of the magnetic field generated by the means for generating the magnetic field, and this figure shows the approach of the means to the measurement cell. It is a good example of the measurement method carried out by the apparatus of the present invention through the modulation of the magnetic field generated by the means for generating the magnetic field, and this figure shows the distance of the means to the measurement cell. It shows the shape of the signal with respect to the difference in the percentage of the resistance component of the impedance after approaching / moving away from the magnetizing means. It is a figure which becomes a good example of the positioning of the detection electrode and the reference electrode with respect to the concentrator in the 1st Example of this invention. A cross section of a measuring cell according to a first embodiment of the apparatus of the present invention is shown, the cross section being along a plane perpendicular to the larger dimension of the at least one concentrator. The details of the cross section shown in FIG. 7a for the at least one concentrator are shown. The details of the cross section shown in FIG. 7a for the at least one detection electrode pair are shown. It is a view from the top of the detail of the 1st Embodiment of the apparatus of this invention, and the said detail is composed of the substrate of the measuring cell. It is a figure seen from the top of the detail configured by the measurement cell of the 2nd Embodiment of this invention. In the second embodiment of the present invention when δ = 40 micron and α = 90 °, the trends of the percentage change of the resistance component of the impedance different from the detection electrode and the reference electrode are shown in (i) plasma. As a function of the equivalent parasite level of the red blood cell sample treated to be paramagnetic (solid symbol and dashed line), and (ii) a healthy red blood cell only sample that provides a signal display due to false positives. In case (dotted line), it is shown. The signal trend for 0.5% equivalent parasitemia at α = 90 ° is shown according to the difference in the height (δ) of the measurement cell. It shows the trend of the ratio of the true signal (0.5% equivalent parasitemia, δ = 40 microns) to the spurious signal for various values α of the angle. It is a figure seen from the top of the detail which is composed of 1 unit measurement cell of the 3rd Example of this invention. It is a view from the top of the detail which is composed of 2 units of the measuring cell of the 3rd Example of this invention. It is a top view of the details composed of one unit of measuring cells of the fourth embodiment of the present invention. It is a figure which looked at the detail of the 3rd and 4th Examples of the apparatus of this invention from the top, and the said detail is composed of the substrate of the measuring cell.

With reference to FIGS. 1, 2, 3, 3a, 3b, and 3c, an embodiment of the apparatus (100) of the present invention for the purpose of quantifying erythrocytes infected with Plasmodium malaria and erythrocytes infected with hemozoin crystals. teeth,
-Measurement cell (1)
Multiple detection electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194'),
-The component to be quantified (3, 3', 3'') is magnetically attracted, and the component is detected by the detection electrode (4, 4', 5, 5', 6, 6', 34, 34', 84', 84, At least one concentrator (10, 10', 10'', 14, 14', 14') configured to be concentrated on 84', 94, 94', 184, 184', 194, 194'). ', 15), the detection electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194. ') Is placed in close proximity to the at least one concentrator (10, 10', 10'', 14, 14', 14'', 15), at least one concentrator (10, 10', 10'). ', 14, 14', 14'', 15),
-At least one for each detection electrode pair (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194'). A reference electrode pair (7,7', 8,8', 9,9', 37, 37', 64, 64', 74, 74', 164, 164', 174'), wherein the reference electrode ( 7, 7', 8, 8', 9, 9', 37, 37', 64, 64', 74, 74', 164, 164', 174') is the at least one concentrator (10, 10'). At least one reference electrode pair (7,7',8,8', 9,9', 37,37) not placed in close proximity to 10'', 14, 14', 14'', 15) ', 64, 64', 74, 74', 164, 164', 174'),
The housing of the detection electrode (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194') and the reference electrode ( 7,7', 8,8', 9,9', 37,37', 64, 64', 74, 74', 164, 164', 174') housings and concentrators (10, 10', 10') '', 14, 14', 14'', 15) the substrate (11) configured for the housing, the first surface (11') facing outward of the cell (1) and the cell. The substrate (11), which has a second surface (11 ″) facing inward of (1),
A first surface (12') facing the outside of the cell (1) and a second surface facing the inside of the cell (1) and facing the first surface (11') of the substrate (11). Support (12), having a surface (12 ″),
To contain at least one spacer element (13, 13') configured to confine the sample and keep the substrate (11) away from the support (12), and to contain the cell (1). Electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194', 7, 7', 8, 8 ', 9, 9', 37, 37', 64, 64', 74, 74', 164, 164', 174) and configured to provide electrical contact between the electronic board (202). Mechanical housing (600)
The measuring cell (1) having the
-An electronic unit (201) connected to the board (202), which generates an input signal, amplifies an output signal, and detects electrodes (4, 4', 5, 5', 6, 6', 34, Measurement of impedance between 34', 84, 84', 94, 94', 184, 184', 194, 194'), reference electrode (7, 7', 8, 8', 9, 9', 37, 37') An electronic unit configured for measuring the impedance between', 64, 64', 74, 74', 164, 164', 174'), or measuring the difference between them, and communicating with the user interface. (201) and
-Means for generating magnetic fields with gradients that can be modulated over time (101, 102, 103) and concentrators (10, 10', 10'', 14, 14', 14'', 15 ), Coupled with
-Separation of the component to be quantified (3, 3', 3'') from the rest of the solution, and-Detection electrode (4, 3', 3'') of the pre-component (3, 3', 3'') to be quantified. 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194') generate a magnetic field capable of causing concentration. And the means that are configured to
-A microfluidic system with pre-administration fluid that performs blood dilution and anticoagulation, as well as transfer into measurement cell (1), integrated with measurement cell (1) and at least partially mechanical. The microfluidic system contained in the housing (600),
-Has a mechanical system configured to vary the angle α formed between the perpendicular to the substrate of the measurement cell from which the blood sample is collected and the gravitational acceleration vector from 0 ° to 180 °.

This time, the microfluidic system
-Means for collecting a blood sample (500) in a cartridge (503) and diluting with a premedication fluid (501), and
-Having a means (504) for moving the diluted blood sample (500) into the measurement cell (1).

Therefore, the patient's blood droplets are placed in contact with the inlet of the microfluidic system so that the analysis can be performed. Thereby, the blood droplets are sucked in, diluted with the premedication fluid and carried into the measurement cell containing the electrode-attached substrate.

The measuring cell may be made by microfabrication techniques on silicon, glass, or other polymeric material coupled with a suitable microfluidic system made of plastic material.

In the first embodiment of the apparatus of the present invention, which is specifically intended for use in the subject of malaria, the means for generating magnetic fields (101, 102, 103) are paramagnetic with respect to the liquid medium in which they are dispersed. For magnetic or diamagnetic components, it has a strength and fine gradient of at least 10 ⁴ A / m of its square module of at least 5 ^* 10 ¹⁴ A ² / m ³ directed at or away from the substrate. Is preferable, it should be possible to generate a magnetic field. In such means (101, 102, 103), for example, the magnetic field generated by the plurality of permanent magnets (101, 102) acts on the target component far away from the substrate (11), thereby causing them. It may be embodied by the magnets (101, 102) positioned to exert sufficient force to counteract the resistance of gravity and Archimedes forces attracting to the surface of the substrate (11).

Referring to FIGS. 4a and 4b, the means for generating a magnetic field (101, 102, 103) are specifically magnetized at right angles to the square plane and arranged in the north pole direction. It is composed of two permanent magnets (101, 102) of NdFeBN52 having a parallelepiped shape and a size of 20 × 20 × 5 mm. Between them, the small size of a mild (mild) ferromagnetic material (eg, mu-metal) that has a magnetic field line on the plane of symmetry of the two magnets (101, 102) and has a high magnetic permeability to concentrate it. There is a thin plate (103). Thereby, on the interface between the small thin plate (103) and air, a symmetric magnetic field and a magnet in which a dominant component is placed in contact with or in close contact with the back surface of the substrate (11) due to a high gradient. It is generated with respect to the separation surface of the magnet (101, 102), which is oriented so as to be orthogonal to the square surface of (101, 102). As described above, since the generated magnetic field does not include the boundary effect of the magnets (101, 102), the components (3, 3', 3'" are formed in a rectangular region having a width of about 2 mm and a height of about 16 mm. It has been confirmed that there is sufficient strength and gradient to capture').

Refer to FIGS. 4a, 4b, and 5, and as described above, the means for generating the magnetic field (101, 102, 103) is the concentrator element (10, 10', 10'', 14, 14'. , 14'', 15) so as to embody the modulation and relative gradient of the magnetic field in the substrate (11), i.e., from the minimum value not exceeding 10% of the saturated magnetic field of the magnetic concentrator to the magnetic concentrator (10, 10). '10'' 14, 14', 14'', 15) are configured to result in a change in magnetic field strength over time to a maximum value that should be higher than the saturated magnetic field. In the first embodiment of the present invention, the magnetic concentrator is made of, for example, Ni, and such modulation is caused by a linear motion of the magnet (101, 102) approaching / moving away from the substrate (11). Can be obtained. When the means for generating the magnetic field (101, 102, 103) is close to the substrate (11), the magnetic field on the Ni concentrator (10, 10', 10'', 14, 14', 14'', 15) However, it saturates its magnetization, which is sufficient to allow capture of the component (3, 3', 3''). As the means for generating the magnetic field (101, 102, 103) move away, the components (3, 3', 3'') become concentrators (10, 10', 10'', 14, 14', 14'', 15'. ) And from the detection electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194'). As such, the magnetic field becomes negligible and the concentrators (10, 10', 10'', 14, 14', 14'', 15) are demagnetized. As already mentioned, the detection electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194', 194', 194', 194', 194', 194', 194', 194', 194' , 194'), the trend of the resistance component of the impedance measured at the end is shown in FIG. At the approaching stage, the positive ΔR increases, assuming components (3, 3', 3'') that have a higher resistance than the resistance of the liquid that develops over the unique capture time _τC . Following a sudden distance from the means for generating the magnetic field (101, 102, 103), the resistance is at a specific time _τR , the detection electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194') return to its original level for desorption of components.

With reference to FIGS. 6, 7a, 7b, and 7c, in the first embodiment of the present invention, each detection electrode pair (4, 4', 5, 5', 6, 6 of the measurement cell (1)) It is assumed that') has a first electrode (4, 5, 6) and a second electrode (4', 5', 6') capable of receiving a first input signal (V ⁺ ). is doing. Each reference electrode pair (7, 7', 8, 8', 9, 9') may receive a second input signal (V ⁻ ) having the opposite polarity to the first input signal (V ⁺ ). The first of the detection electrode pairs (4, 4', 5, 5', 6, 6') at the common point from which the output signal (Out) is taken out from the first electrode (7, 8, 9) that can be formed. It has a second electrode (7', 8', 9') connected to a second electrode (4', 5', 6'). By overtly modifying the electronic unit (201), the role of the electrode is reversed, the common point of the second detection electrode (4', 5', 6') and the second reference electrode (7', 8', 9'. The shared point of') is used as an input signal, the first electrode (4, 5, 6) of the detection electrode pair is used as the first output signal, and the first electrode (4, 5, 6) of the reference electrode pair is used. It is also possible to use 7, 8 and 9) as the second output signal. In any case, the electronic unit (201) will be embodied to give the user the difference between the impedance of the detection electrode pair and the impedance of the reference electrode pair in a properly processed form.

In the first embodiment of the present invention, the concentrator (10, 10', 10'') suitable for diagnosing malaria is made of a ferromagnetic material such as Ni, Fe, Co, NiFe, CoFe and has the largest dimensions. Has the shape of a parallelepiped extending at right angles to the plane represented in FIG. 3a. The dimensions of the concentrator (10, 10', 10'') and the detection electrodes (4, 4', 5, 5', 6, 6) to ensure a sufficient concentration factor and obtain a sufficient signal-to-noise ratio. ') Dimension should be within the range listed in Table 1.

In Table 1, h _F is the smallest dimension of the base of the concentrator, w _F is the largest dimension of the base of the concentrator, and d _F is the distance between one concentrator and an adjacent concentrator. h _E is the smallest dimension of the base of the detection electrode, w _F is the largest dimension of the base of the detection electrode, and d _E is the distance between two adjacent electrodes in the same concentrator.

The first row of Table 1 shows the dimensional ranges of concentrators and detection electrodes required for the correct detection of plasmodium-infected erythrocytes (i-RBC), while the second row of Table 1 shows free hemozoin. (HC) Dimensional ranges of concentrators and detection electrodes required for correct detection of crystals are shown.

The substrate (11), and thus the same cell (1) of the invention, the structure of the detection electrodes (4, 4', 5, 5', 6, 6'), and the reference electrode (7, 7', 8, 8'). , 9, 9') structure can be reproduced in four zones where the substrate (11) is divided, each zone divided into two square zones with a base equal to 2 mm and a height equal to 4 mm. Has been done. The width of 2 mm is commensurate with the expansion of the high magnetic field produced by the magnet in the direction perpendicular to the plane of symmetry. The left part carries an electrode on a concentrator and is centered on the plane of symmetry of the magnet described, while the right part has only a reference electrode for subtraction of the common mode signal. Subdivision of the effective area into several regions with unrelated readings allows for an increase in the ratio of the impedance change caused by one component attached on the detection electrode to the overall impedance between the electrodes. Therefore, the signal-to-noise ratio is improved when a low-concentration component is detected. Each zone requires an output contact to the amplifier from which the output signal (Out) will be emitted, while all input signals (V ⁺ ) and (V- ⁾ to the detection and reference electrodes have two contacts. Since only the number of contacts is required, the minimum number of contacts formed on the chip is equal to 4 + 2 = 6. Three separate contacts can be used per zone to minimize the effects of short circuits that can occur between the electrodes during manufacturing. As a result, the total number of contacts is 12.

Referring to FIGS. 8 and 9, in the second embodiment of the apparatus of the present invention, which is also specifically intended for use in malaria, the apparatus (100) is the only difference in the configuration of the measuring cell (1). It is assumed to have all the same components as. In a second embodiment of the invention, the measurement cell (1) is actually a cylindrical ferromagnetic concentrator matrix (14, 14', uniformly dispersed on the substrate (11) according to a square grid. 14''). Alternatively, the concentrator matrix may be arranged according to a hexagonal grid that maximizes filling. In FIG. 9, specifically, six detection electrode pairs (34, 34') and six reference electrode pairs (37, 37') are shown. The first electrode (34) of each detection electrode pair (34, 34') is configured to receive the first input signal (V ⁺ ) via the first connection locus (44). Is connected to the first input. The electrodes may be parallel, as shown in FIG. 9, to take advantage of the experimentally detected capture trends at the edge of the cylinder, thereby maximizing the occupancy of sensitive areas on the electrodes. Alternatively, it may have a ring shape as seen in the examples described below. The first electrode (37) of each reference electrode pair (37, 37') is configured to receive a second input signal (V− ⁾ via a second connection locus (47). It is connected to the second input. Similarly, the second electrode (34') of each detection electrode pair (34, 34') is connected to the node from which the output signal (Out) is emitted through the third connection locus (44'). The second electrode (37') of each reference electrode pair (37, 37') is connected to the node from which the output signal (Out) is emitted through the fourth connection locus (47').

On the first connection locus (44), the second connection locus (47), the third connection locus (44'), and the fourth connection locus (47'), an insulating layer (40) is placed for each locus. , 40', 50, 50'), and the insulating layer (40, 40', 50, 50') is provided with the connection locus (44, 44'so that the effect of this impedance is negligible. , 47, 47') has a dielectric constant and thickness that makes the impedance very high.

Due to the configuration of the concentrator envisioned by the second embodiment, the concentration factor obtained can be even higher than the concentration factor obtained for the first embodiment, at least at α = 0. .. To this end, the dimensions of the concentrator (14, 14', 14'') and the dimensions of the detection electrodes (34, 34', 35, 35') should be within the ranges listed in Table 2. Is preferable.

In Table 2, h _F is the height of the concentrator, w _F is the diameter of the base of the concentrator, and d _F is the distance between one concentrator and an adjacent concentrator. h _E is the smallest dimension of the base of the detection electrode, w _F is the largest dimension of the base of the detection electrode, and d _E is the first finger of the detection electrode and the second finger of the detection electrode. Is the interval with.

Table 2 shows the dimensional ranges of concentrators and detection electrodes required for the correct detection of both erythrocytes (i-RBC) infected with Plasmodium and free hemozoin crystals (HC).

が得られる。図８を参照すると、本発明の第２の実施例でも、基板（１１）、それにより、検出電極（４、４’、５、５’、６、６’）の構造、及び基準電極（７、７’、８、８’、９、９’）の構造が、基板（１１）が分けられている４つのゾーンに再現され得、各ゾーンが、左パート、すなわち第１のユニットがコンセントレータ上に電極を担持し、記載の磁石の対称面（８００）を中心とする一方、右パート、すなわち第２のユニットが、コモンモード信号の減算に基準電極のみを有する、２つの長方形ゾーンに分けられている。 With such dimensions, under optimal spacing conditions, assuming a straight electrode length L, shown in FIG. 9, equal to 6 μm, and a capture efficiency of 100%, a geometric concentration factor of about 400.

Is obtained. Referring to FIG. 8, also in the second embodiment of the present invention, the substrate (11), thereby the structure of the detection electrode (4, 4', 5, 5', 6, 6'), and the reference electrode (7). , 7', 8, 8', 9, 9') can be reproduced in four zones where the substrate (11) is divided, with each zone being the left part, i.e. the first unit on the concentrator. The right part, i.e., the second unit, is divided into two rectangular zones having only a reference electrode for the subtraction of the common mode signal, while carrying electrodes on the above and centered on the plane of symmetry (800) of the described magnet. ing.

With reference to FIGS. 13, 14, and 16, in a third embodiment of the apparatus of the present invention, which is also specifically intended for use in malaria, the apparatus (100) is a measuring cell (100) as follows. It is assumed to have all the same components as above, which differ in terms of the configuration of 1).

As seen in the first and second embodiments, the substrate (11), thereby the structure of the detection electrode (84, 84', 94, 94'), and the reference electrode (64, 64', 74, 74'). ) Can be reproduced in four zones (301, 302, 303, 304) where the substrate is divided. The minimum number of contacts formed on the chip is always equal to 4 + 2 = 6, in which 4 output contacts (1 per zone) and 2 input contacts (all detection electrodes and reference). There is one for the first input signal (V ⁺ ) for the electrodes and one for the second input signal (V ⁻ ) for all detection and reference electrodes. However, as already mentioned for the first and second embodiments, three separate contacts (Out, V ⁺ ,) per zone are used to minimize the effects of possible short circuits between the electrodes during manufacturing. V- ⁾ can be used. As a result, the total number of contacts is 12.

Unlike the first and second embodiments, at least one part of the substrate (11) is centered on the plane of symmetry (801) of the means for generating the magnetic field (101, 102, 103), said part. ,
-The detection electrode and the reference electrode (84, 84', 94, 94', 64, 64', 74, 74') are usually alternately mated so as to receive the same magnetic field, fluctuation, and drift. More generally, the combined detection electrode (84, 84', 94, 94') and the reference electrode (64, 64', 74, 74') and the reference electrode (64, 64', 74, 74').
-Supports a concentrator (10, 10', 10'', 14, 14', 14'') in close proximity to the detection electrode (84, 84', 94, 94').

For the purposes of this specification, the term "intergitated" means that one or two strips are staggered and closely packed. When the detection electrode (84, 84', 94, 94') and the reference electrode (64, 64', 74, 74') are alternately fitted, the background subtraction is improved, thereby being quantified. Obtain a more accurate measurement of the impedance change corresponding to the component of. In FIG. 13, specifically, 6 detection electrode pairs (84, 84', 94, 94'), 6 reference electrode pairs (64, 64', 74, 74'), and 6 concentrators. (14, 14', 15) are shown. As seen in the second embodiment, the concentrator is ferromagnetic and has a cylindrical shape (14, 14', 15). The electrodes (64, 64', 74, 74', 84, 84', 94, 94') and concentrators (14, 14', 15) are uniformly dispersed on the substrate according to a rectangular grid. The concentrator (14, 14', 15) is placed in close proximity only to the detection electrode (84, 84', 94, 94') and in close proximity to the reference electrode (64, 64', 74, 74'). Not. The detection electrode (84, 84', 94, 94') and the reference electrode (64, 64', 74, 74') are arranged in alternating double strips. More specifically, in each row, both of the two detection electrode pairs (84, 84', 94, 94') are the first reference electrode pair (74, 74') and the second reference electrode. No reference electrode (74, 74', 64, 64') is inserted between the pair (64, 64') and the two detection electrode pairs (84, 84', 94, 94'). Not sandwiched. More generally, the measurement cell has at least a first detection electrode pair (84, 84', 184, 184') and a second detection electrode pair (94, 94', 194, 194'). , Both of the detection electrode pairs (84, 84', 184, 184', 94, 94', 194, 194') are the first reference electrode pair (74, 74', 174, 174') and the second. It is sandwiched between the reference electrode pair (64, 64', 164, 164'), and the first detection electrode pair (84, 84', 184, 184') and the second detection electrode pair (94, 94'). No reference electrode (74, 74'174, 174', 64, 64', 164, 164') is inserted between the reference electrode (', 194, 194'). The spatial structure and dispersion of the detection electrode (84, 84', 94, 94') and the spatial structure and dispersion of the reference electrode (64, 64', 74, 74') are as shown in FIG. It forms a unit (700) that can be reproduced to form a second unit (701) and many other identical units. In this way, a layout is created, in which the reference electrode (64, 64', 74, 74') and the detection electrode (84, 84', 94, 94') are snugly alternated and thus. It promises better subtraction of false variability and, as a result, increased sensitivity of tests performed using the device (100) of the present invention.

As a result, the first electrode (84', 94, 184', of each detection electrode pair (84, 84', 94, 94', 184, 184', 194, 194') of the measurement cell (1), 194) is connected to the first input V ⁺ , while the first electrode (64,74') of each reference electrode pair (64, 64', 74, 74') is the second input. It is connected ^to V-. The second electrode (84,94') of each detection electrode pair (84, 84', 94, 94') and the second of each reference electrode pair (64, 64', 74, 74'). Electrodes (64', 74) are connected to the node from which the output signal (Out) is emitted. The electrodes (64, 64, 74, 74', 84, 84', 94, 94') are ring-shaped. Specifically, one end of each pair (64, 64', 74, 74', 84, 84', 94, 94') of electrodes (64, 74, 84, 94) has two open ends. Located in a concentric ring. The other (64', 74') of the electrodes (64', 74', 84', 94') of each pair (64, 64', 74, 74', 84, 84', 94, 94'). , 84', 94'), instead consists of a straight stretch and an open ring surrounding the straight stretch. This extension is configured to enter the inner ring at the end of the first electrode (64, 74, 84, 94) of the pair, and the open ring surrounding the straight extension is the first of the pair. It is configured to enter the space between the inner ring and the outer ring of the electrodes (64, 74, 84, 94) to be formed. Referring to FIG. 15, in a fourth embodiment of the apparatus of the present invention, which is also specifically intended for use in malaria, it is assumed that the apparatus (100) has all the same components as above. The concentrators (14, 14', 15) have the same geometry (ie, cylindrical shape) as the concentrators of the third and second embodiments. The measurement cell (1) has the same relative positioning between the above-mentioned detection electrode and the reference electrode (164, 164', 174, 174', 184, 184', 194, 194') as in the third embodiment. In the fourth embodiment, the reference electrode (164, 164', 174, 174') and the detection electrode (184, 184', 194, 194') are closely and alternately fitted, specifically, staggered two. It is characterized by the same layout as in the third embodiment, arranged in heavy strips. The only difference is the geometry of the electrodes. In the fourth embodiment, one end of the electrodes (164, 174, 184, 194) of each pair (164, 164', 174, 174', 184, 184', 194, 194') is open ring. It is in. The other one (164', 174') of the electrodes (164', 174', 184', 194') of each pair (164', 164', 174, 174', 184, 184', 194, 194'). , 184', 194'), instead consist of a closed ring configured to enter the open ring of the pair's first electrode (164, 174, 184, 194). Further, in the fourth embodiment, the substrate (11), thereby the structure of the detection electrode (184, 184', 194') and the structure of the reference electrode (164, 164', 174, 174') are separated by the substrate. It can be reproduced in the four zones (301, 302, 303, 304). To minimize the effects of short circuits that can occur between the electrodes during manufacturing, three separate contacts (Out, V ⁺ , V- ⁾ can be used per zone with a total number of contacts equal to 12. .. However, the minimum number of contacts formed on the chip is 6, four output contacts (one per zone) and two input contacts (first for all detection and reference electrodes). One for the input signal (V ⁺ ) and one for the second input signal (V ⁻ ) for all detection and reference electrodes).

(Illustration)
The embodiments described below relate to the characterization of the device according to the second embodiment above, specifically intended for use in malaria. The substrate with the structure depicted in FIG. 8 defines the placement of Ni concentrators (40 microns in diameter, 20 microns in height) arranged according to a hexagonal grid with an intercenter spacing of 160 microns. The electrodes are ring-shaped and are made of gold with a thickness of 300 nm, a width of 3 microns and an interval of 3 microns. The external electrode has an outer diameter of 40 microns, specifically on the edge of the cylinder, the maximum value of the magnetic field and the maximum value of its gradient, in order to maximize the possibility of capturing components in the electrode. It is completely superposed on the Ni concentrator. An insulating layer of SiO ₂ is sandwiched between the electrode and the upper surface of the concentrator with a thickness of 3 microns. The distance between the substrate and the support is determined by the polymer container ring (50) having a thickness of 40 to 500 microns and varying.

A sample droplet containing the component to be analyzed is administered to a support on which the confinement ring is assembled and first placed horizontally upwards. The substrate is lowered until the confinement ring is pushed, resulting in a seal that allows the fluid cell to be defined. The cell is housed in a mechanical device that allows variation in the angle α from 0 to 180 °, which is a vertical line to the gravitational acceleration vector with respect to the surface of the substrate. The motorized linear motion makes it possible to controlally move the magnet closer to the substrate and away from it, thereby measuring the resistance change proportional to the concentration of the components. The NdFeB magnet configured as shown in FIG. 1 is close to the surface of the substrate towards the measurement cell, i.e. the module of the square module and the gradient are ^{6 *} 105 Am ^-1 , 7 respectively. ^* 10 ¹⁴ A A magnetic field H can be generated on the plane of symmetry of two facing magnets, which is equal to A ² m ^-3 , at a distance of 0.5 mm from the surface of the magnet.

In a specific application for malaria diagnosis, the components involved in the paramagnetic properties of plasma are hemozoin crystals and erythrocytes infected with Plasmodium including several hemozoin crystals. Both hemozoin crystals and red blood cells act as insulators to the input voltage signals applied to them in the range of several MHz, such as the input voltage signals considered for impedimetric detection. Hemozoin is 4.1 ^* 10 ^-4 S. I. It has an absolute positive volume magnetic sensitivity, which is equal to the unit, but [M. Giacometti et al. APPLIED PHYSICS LETTERS 113, 203703 (2018)], infected erythrocytes, as a whole, have more diamagnetic behavior. However, the difference in susceptibility between infected erythrocytes and plasma is lower than the volume susceptibility of hemozoine crystals, but sufficient to bring about their capture at the appropriate gradient of the applied magnetic field H. The volume susceptibility is not more negative than the volume magnetic susceptibility of plasma so that it is about 8 ^{* 10-6} . The difference in susceptibility between the two components distinguishes them and allows the values of the magnetic field gradient and angle α to be appropriately selected based on an approximation of the forces involved.

により、血球と血漿との感受率差Δχ＝１．８^＊１０^－６［Ｋ．Ｈａｎ ａｎｄ Ａ． Ｂ． Ｆｒａｚｉｅｒ、 Ｊ． Ａｐｐｌ． Ｐｈｙｓ． ９６（１０）、 ５７９７頁（２００４）］と仮定すると、α＝０°の場合のバランスＦ_ｇｂに必要なＨ^２勾配の値は、１^＊１０^１５Ａ^２ｍ^－３程度になる。 Assuming that the red blood cell volume V _RBC = 9.1 ^{* 10-11} cm ^-3 , blood cell density ρ _RBC = 1.15 gcm ^-3 , and plasma density ρ _P = 1.025 g cm ^-3 , the weight force and archimedes applied to one blood cell. The sum of the forces and F _gb = (ρ _RBC −ρ _P ) V _RBC g (Fig. 3a) gives a result equal to 1.1 ^{* 10-13} N. Equation of magnetic force applied to superparamagnetic particles

Therefore, the difference in susceptibility between blood cells and plasma Δχ = 1.8 ^{* 10-6} [K. Hand and A. B. Frazier, J.M. Apple. Phys. Assuming 96 (10), p. 5797 (2004)], the value of the H2 gradient required for the balance F _gb when α = 0 ° is about ^{1 *} 10 ¹⁵ A ² m ^-3 .

Similar estimates can be made for one plasma suspended hemozoin crystal. Assuming that the average volume V _HC = 2.2 ^* 10-14 cm ^-3 , the density ρ _HC = 1.15 gcm ^-3 , and the difference in susceptibility rate ^Δχ = 4.1 ^* 10 ^-4 for plasma, the balance in hemozoin It can be obtained that the value of the H ² gradient required for F _gb is about 17 ^* 10 ¹³ A ² m ^-3 .

Therefore, from these estimates, at an angle α = 0, the H2 gradient (magnet is 0.5 mm thick back surface of a substrate) provided by a particular magnet that is considered when the magnetic force is antiparallel to the resultant force of gravity and Archimedes forces. The result is that 7 ^* 10 ¹⁴ A ² m ^-3 ) on the surface of the substrate's electrodes when sitting on the substrate can attract hemozoin crystals rather than malaria-infected erythrocytes.

In experiments performed at an angle α = 0 in a suspension of hemozoin crystals at plasma dilution 1:10 with PBS to simulate the expected blood dilution in the diagnostic test, in fact, to a hemozoin concentration equal to 1 ng / ml. , Showed the possibility of measuring the net change in resistance between the electrodes of the substrate, which can be distinguished from noise. Assuming that there are about 18 hemozoin crystals in the malaria-infected red blood cells, the detected concentration is 0.2% (sick red blood cells and health, just typical of patients with malaria febrile attacks. Corresponds to malaria (percentage of red blood cells).

Measurements of NaNO ₂ treated erythrocyte samples to induce hemoglobin transformation into paramagnetic methemoglobin, which allows an erythrocyte model of malaria infection to be obtained under the same conditions (angle α = 0). , Showed that there is no signal distinguishable from noise. Even when treated red blood cells (t-RBC) have a difference in magnetic sensitivity to plasma, which is twice the magnetic sensitivity of infected blood cells (Δχ = 3.6 ^{* 10-6} ), [Nam, Jeonghun. , Hui Hung, Hyunjung Lim, Plasmaung Lim, Sehyun Shin. Analytical Chemistry 85, n. 15, 7316-23 (2013)], to fully prove that there is no capture, and thus no electrical signal, of any non-idealism or deviation from the values listed for the properties of red blood cells. The H2 gradient (5 ^* 10 ¹⁴ A ² m ^-3 ) required to balance gravity and buoyancy is the H2 gradient (7 ^* 10 ¹⁴ A ² m ^-3 ) brought about by the magnet. It's very similar.

The situation changes at an angle α close to 90 °, where the weight and buoyancy components that oppose the magnetic force are virtually zero. Since there is no actual threshold anymore, both hemozoin and erythrocytes are attracted to the substrate by the macroscopic magnetic field gradient, thereby concentrating on the concentrator by the local gradient during their downward sliding motion.

In hemozoin, the value of the signal detected as a function of concentration does not vary significantly because it is already very effective when the attraction and concentration are α = 0. Therefore, a detection limit very similar to the detection limit found at α = 0 of about 1 ng / ml is obtained at α = 90 °.

In treated red blood cells, instead, there is a more relevant signal that allows a detection limit (LOD: Limit Of Detection) of around 0.005%, which corresponds to about 250 parasites per μl of blood. can get. At frequencies of 1 MHz input signals (V ⁺ and V- ⁾ , erythrocytes have an insulating behavior, so that the change in the measured resistance component of the ΔR / R impedance is the volume fraction it occupies on the concentrator electrode. Is proportional to. It reveals how a larger volume of red blood cell hemozoin can lead to a wider range of signals.

FIG. 10 shows the percentage change in resistance measured as a function of the equivalent parasitemia level of the sample according to the distance of the magnet from the substrate, i.e. during the release of red blood cells attached to the electrodes, with dots and solid lines. Given that the diagnostic test involves diluting the patient's blood in PBS (1:10 vol-vol), the sample is less concentrated, which may correlate with parasitemia, as follows: Consists of a suspension of NaNO ₂ treated erythrocytes in a solution of plasma and PBS (1:10). Since hematocrit is about 4% in 1:10 diluted blood, the equivalent parasitemia is χ / 4, where χ is the percentage volume fraction of t-RBC in the sample.

Also in FIG. 10, the dotted line corresponds to the ΔR / R signal measured in 0.4% hematocrit plasma and a sample of untreated red blood cells resuspended in PBS (1:10 vol-vol). This signal corresponds to some false positives and some to false fluctuations induced by the movement of the magnet, but below that it forms the lower limit of the signal from which no significant measurements can be made. .. As can be seen, the measured ΔR / R signal follows a nearly linear trend of sample parasitemia over a long period of more than 30 years, thereby allowing quantification of parasitemia itself. .. The detection limit resulting from the intersection of the two curves is about 0.05% per μl of blood, or about 250 parasites, which corresponds to the LOD of the current rapid diagnostic test for malaria. However, in contrast, the tests covered by this patent application allow for a quantitative estimation of parasitemia, which is useful in the diagnostic and monitoring stages of the disease following drug treatment.

The effect of angle α on the detection limit has been studied in experiments performed at angles of 75 °, 90 °, and 105 °, as shown in FIG. This is the ΔR / R signal measured in a 0.5% equivalent parasitemia t-RBC sample and 0.4% hematocrit plasma and PBS (1:10 vol-vol) corresponding to false positives. ) Shows the ratio to the ΔR / R signal measured in the sample of untreated red blood cells resuspended in. This ratio, which may correspond to the true / non-specific signal ratio of this test, is maximum at 90 °, but decreases at larger or smaller angles. At 75 °, gravity not only reduces false-positive signals, but can also accommodate signals that have been processed with true signals, and as a result, there is still a component that opposes the magnetic force so that the ratio is reduced. .. At 105 °, gravity contributes to directing all healthy blood cells and blood cells to be treated towards the electrodes of the substrate so that not only the true signal but also the non-specific signal increases. However, in this case, the signal-to-noise ratio also decreases. Finally, the analysis performed shows that a 90 ° angle is preferred to maximize the ratio of the true signal to the non-specific signal.

The height (δ) of the fluid measurement cell, defined by the thickness of the spacer element, needs to be optimized by itself to increase the ratio of the true signal to the non-specific signal. FIG. 11 shows the amplitude of the current signal measured after the second distant movement from the magnet for cell heights of 40, 80, and 500 microns. As you can see, the net signal decreases with increasing thickness, despite the fact that there are more blood cells in the cell. In the example shown here, the measurement is initiated by anchoring to the substrate within the normal time (3 minutes) required to close the cell, providing electrical contact, stabilizing the signal and closing the magnet. The sample is loaded with the cell horizontal (α = 0) so that the blood cells have time to do so. Even with δ = 500 microns, given the fact that a normal precipitation rate of 2 microns / sec, the thickness of at least 360 microns from the substrate is empty with no blood cells. This means that when the samples approach, the blood cells are farther apart than in the case of δ = 40 microns, which means that their capture during sliding is less effective. Therefore, this analysis shows that according to the protocol adopted in this example, the best condition for increasing the signal corresponds to δ = 40 microns.

The increase in cell thickness, and thereby the increase in the number of blood cells contained therein, can be leveraged instead by proper agitation, if the initial erythrocyte sedimentation rate of blood cells is avoided, or by initially placing the device at an angle α = 180 °. May be. In this way, it may be possible to concentrate blood cells in close proximity to the substrate and utilize gravity-induced sliding motion to keep healthy red blood cells away from the concentrator and instead capture the affected red blood cells.

Finally, you should see how a highly dynamic signal becomes rich in information that makes it possible to distinguish the nature of the signal itself. At α = 90 °, δ = 40 microns, the dynamic signal following the approach of the magnet, which corresponds to the capture time _τC , is a false positive signal corresponding to a sample of 4% hematocrit with healthy (untreated) blood cells. In the case of, there is a value of 80 seconds, and in the case of a sample of treated red blood cells having a volume fraction corresponding to 0.5% parasitemia, there is a value of 150 seconds. This difference also remains for the release times _τR of 20 seconds and 60 seconds, respectively. Since the specific mechanics are known, it is possible to identify false impedance changes, for example by false positives in this system or by other variations.

Claims (17)

A device (100) for quantifying biological components (3, 3', 3'') in a fluid.
-Measurement cell (1)
Multiple detection electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194'),
The component (3, 3', 3'') to be quantified is magnetically attracted, and the component is attracted to the detection electrode (4, 4', 5, 5', 6, 6', 34, 34', 84'. , 84', 94, 94', 184, 184', 194, 194') at least one concentrator (10, 10', 10'', 14, 14', 14') configured to assemble. , 15), and the detection electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194'. ) Is placed in close proximity to the at least one concentrator (10, 10', 10'', 14, 14', 14'', 15), at least one concentrator (10, 10', 10'', 14, 14', 14'', 15),
-At least one reference electrode pair (7, 7', 8, 8) arranged so as not to be close to the at least one concentrator (10, 10', 10'', 14, 14', 14'', 15). ', 9, 9', 37, 37', 64, 64', 74, 74', 164, 164', 174'),
The detection electrode (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194'), the reference electrode ( 7, 7', 8, 8', 9, 9', 37, 37', 64, 64', 74, 74', 164, 164', 174'), and the concentrator (10, 10', 10' A substrate (11) for accommodating', 14, 14', 14'', 15), the first surface (11') facing the outside of the cell (1), and the cell (1). Substrate (11) with a second surface (11 ″) facing inward,
A first surface (12') facing the outside of the cell (1) and a second surface (12 ″) facing the inside of the cell (1) of the substrate (11). A support (12) having a second surface (12 ″) facing the second surface (11 ″),
At least one spacer element (13, 13') that traps the sample and separates the substrate (11) from the support (12), and the electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194, 194') and the mechanical of the cell (1) that makes electrical contact between the electronic board (202). Housing (600)
With the measurement cell (1) and
-An electronic unit (201) connected to the board (202), which generates an input signal, amplifies an output signal, and detects electrodes (4, 4', 5, 5', 6, 6', Impedance between 34, 34', 84, 84', 94, 94', 184, 184', 194, 194'), said reference electrode (7,7', 8,8', 9,9', 37, An electronic unit (201) configured for measuring impedances between 37', 64, 64', 74, 74', 164, 164', 174'), or their differences, and communicating with a user interface. )When,
-In the device (100) having means (101, 102, 103) for generating a magnetic field having a time variation intensity and a time variation gradient.
The means are configured to create time variability up to the maximum intensity of the magnetic field above the saturated magnetic field of the concentrator (10, 10', 10'', 14, 14', 14'', 15). Also, the means, in collaboration with the concentrator (10, 10', 10'', 14, 14', 14'', 15),
-Separation of the quantified component (3, 3', 3'') from the rest of the solution,
-The detection electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94'of the component (3, 3', 3'') to be quantified. ', 184, 184', 194, 194'), the apparatus (100), characterized in that it is configured to generate a magnetic field that can cause aggregation on. The measurement cell (1) is a pair of detection electrodes (4, 4', 5, 5', 6, 6', 34, 34', 84, 84', 94, 94', 184, 184', 194. , 194') with respect to at least one reference electrode pair (7, 7', 8, 8', 9, 9', 37, 37', 64, 64', 74, 74', 164, 164', 174. ') The apparatus (100) according to claim 1. The measurement cell (1) is fixed at an inclined position such that the angle between the normal of the cell (1) with respect to the substrate (11) and the gravitational acceleration vector is 0 ° to 180 °. The device (100) according to claim 1 or 2. Claim 1 or 2 having a mechanical system configured to vary the angle between the normal of the measurement cell (1) with respect to the substrate (11) and the gravitational acceleration vector from 0 ° to 180 °. The device (100) according to. The apparatus according to any one of claims 1 to 4, comprising a microfluidic system comprising a pre-administration fluid (501) configured for dilution of a biological sample and transfer within said measurement cell (1). 100). The microfluidic system
-Means for collecting the fluid sample (500) and diluting it with the pre-administered fluid (501) on the cartridge (503).
5. The apparatus of claim 5, comprising means (504) for transferring the diluted fluid sample (500) within the measurement cell (1). The device according to claim 5 or 6, wherein the microfluidic system is configured for anticoagulation of the biological sample, wherein the biological sample contains blood. The device of claim 4 or 5, wherein the microfluidic system is integrated with the measurement cell (1) and the microfluidic system is at least partially contained in the mechanical housing (600). The at least one spacer element (13, 13') is in the form of a ring appropriately shaped to carry the component (3, 3', 3'') into the zone of the electrode. The apparatus (100) according to any one of 1 to 8. The means (101, 102, 103) for generating a magnetic field said at the substrate (11) by the linear motion of approach / retreat of the means (101, 102, 103) for generating a magnetic field. The device (100) according to claim 9, which is configured to create a time variation of the magnetic field. The means (101, 102, 103) for generating a magnetic field are
-Two permanent magnets (101, 102), a parallelepiped with a square or polygonal base, in the form of a parallelepiped magnetized at right angles to the base.
-A mild ferromagnetic thin plate (103) arranged between the two permanent magnets (101, 102) between the bases of the magnets having the same polarity, the two magnets (101). , 102) The apparatus (100) according to any one of claims 1 to 10, further comprising a thin plate (103) configured to concentrate magnetic field lines in the plane of symmetry of the assembly. .. -The first electrode (34) of each pair (34, 34') of the detection electrodes of the measurement cell (1) is connected to the first input by the first connection trajectory (44).
-The first electrode (37) of each reference electrode pair (37, 37') is connected to the second input by a second connection trajectory (47).
-A second electrode (34') of each pair of detection electrodes (34, 34') is connected to a node, from which an output signal (Out) is delivered by a third connection trajectory (44'). Emitted,
-The second electrode (37') of each reference electrode pair (37, 37') is connected to the node, from which the output signal (Out) is delivered by the fourth connection trajectory (47'). Emitted,
An insulating layer (40, 40', 50, 50') is arranged on each track of the connecting track (44, 44', 47, 47'), and the insulating layer (40, 40', 50, 50') is arranged. ') Have a dielectric constant and a thickness such as to increase the impedance between the connecting trajectories (44, 44', 47, 47'), thereby making the effect of the impedance negligible. The device (100) according to any one of 2 to 11. The substrate (11) is divided into at least two parts, the first part being the detection electrode (4, 4', 5, 5', 6, 6', 34, 34') and the detection electrode (4, 4', 5', 6, 6', 34, 34'). The concentrator (10, 10', 10'', 14, 14', 14'') in close proximity to 4, 4', 5, 5', 6, 6', 34, 34') is carried and the magnetic field is applied. Is centered on the plane of symmetry of the means (101, 102, 103) for generating, and the second part is the reference electrode (7,7', 8,8', 9,9', Only 37,37') is carried so that the reference electrode (7,7', 8,8', 9,9', 37,37') is the detection electrode (4, 4', 5, 5'). , 6, 6', 34, 34'), the apparatus (100) according to any one of claims 2 to 12, which receives a magnetic field lower than that of (6, 6', 34, 34'). At least one part of the substrate (11) is centered on the plane of symmetry (801) of the means (101, 102, 103) for generating a magnetic field, and the part is
-The detection electrodes (84, 84', 94, 94') and the reference electrodes (64, 64', 74, 74') are alternately mated so as to be exposed to the same magnetic field, fluctuation and drift on average. With the detection electrode (84, 84', 94, 94') and the reference electrode (64, 64', 74, 74'),
-From claim 2, which carries the concentrator (10, 10', 10'', 14, 14', 14'') in the vicinity of the detection electrode (84, 84', 94, 94'). 11. The apparatus (100) according to any one of up to 11. The first electrode (4, 5, 6, 34) of the at least one detection electrode pair (4, 4', 5, 5', 6, 6', 34, 34'), and the at least one. The second electrode (4', 5', 6', 34') of the two detection electrode pairs (4, 4', 5, 5', 6, 6', 34, 34') is 10 to 10 to. 12. The apparatus (100) of claim 12, which has a base of 300 nm and a rectangular cross section as high as 1-3 μm. The first electrode (4, 5, 6, 34) of the at least one detection electrode pair (4, 4', 5, 5', 6, 6', 34, 34') and the at least one. The distance between the detection electrode pair (4, 4', 5, 5', 6, 6', 34, 34') and the second electrode (4', 5', 6', 34') is The apparatus (100) according to claim 15, which is 1 to 5 μm. The at least one concentrator (14, 14', 14'', 15) has a cylindrical shape, and the diameter of the base surface of the concentrator (14, 14', 14'', 15) is 10 to 50 μm. , The height of the concentrator (14, 14', 14'', 15) is 10 to 50 μm, and the distance between the concentrators (14, 14', 14'', 15) is 50 to 150 μm. , The apparatus (100) according to any one of claims 12 to 17.

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